Hypothalamic GRF agonists

ABSTRACT

Synthetic peptides are provided which are extremely potent in stimulating the release of pituitary GH in animals and which have the formula: H--R 1  --Ala--Asp--Ala--Ile--Phe--Thr--R 8  --Ser--R 10  --Arg--R 12  --R 13  --Leu--R 15  --Gln--Leu--R 18  --Ala--Arg--Lys--Leu--Leu--R 24  --R 25  --Ile--R 27  --R 28  --Arg--Gln--Gln--Gly--Glu--R 34  --Asn--Gln--Glu--R 38  --R 39  --R 40  --Arg--R 42  --R 43  --Leu--Y wherein R 1  is Tyr, Met, Leu, D--Tyr, D--His or His; R 8  is Ser or Asn; R 10  is Tyr or D--Tyr; R 12  is Arg or Lys; R 13  is Ile or Val; R 15  is Gly or D--Ala; R 18  is Tyr or Ser; R 24  is His or Gln; R 25  is Glu or Asp; R 27  is Met, Ala, Nle, Ile, Leu or Val; R 28  is Asn or Ser; R 34  is Arg or Ser; R 38  is Gln or Arg; R 39  is Arg or Gly; R 40  is Ser or Ala; R 42  is Phe or Ala; R 43  is Asn or Arg; and Y represents the carboxyl moiety of the amino acid residue at the C-terminus and is the radical --COOR,--CRO,--CONHNHR, --CON(R)(R&#39;) or --CH 2  OR, with R and R&#39; being lower alkyl, fluoro lower alkyl or hydrogen, or a fragment thereof extending from the N-terminus in a continuous sequence at least to the residue in position 29, which fragment is biologically active to cause the release of GH from the pituitary, or a nontoxic salt of such peptide or peptide fragment. The peptides are analogs of human GRF and contain at least two but less than all of the following residues: R 8  is Asn, R 12  is Lys, R 13  is Val, R 18  is Ser, R 24  is Gln, R 25  is Asp, R 28  is Ser.

This invention was made with Government support under Grant No. AM-26741 awarded by the National Institutes of Health. The Government has certain rights in this invention.

This application is a continuation-in-part of our copending application Ser. No. 488,748 filed Apr. 26, 1983 now U.S. Pat. No. 4,595,676.

The present invention relates to peptides having influence on the function of the pituitary gland in humans and other animals. In particular, the present invention is directed to a peptide which promotes the release of growth hormone by the pituitary gland.

BACKGROUND OF THE INVENTION

Physiologists have long recognized that the hypothalamus controls all the secretory functions of the adenohypophysis with the hypothalamus producing special polypeptides which trigger the secretion of each pituitary hormone. Hypothalamic releasing factors have been characterized for the pituitary hormones thyrotropin and prolactin (the tripeptide TRF), for the pituitary gonadotropins luteinizing hormone and follicle stimulating hormone (the decapeptide LRF, LH-RH or GnRH) and for the pituitary hormones β-endorphin and adrenocorticotropin (the 41-amino acid polypeptide CRF). An inhibitory factor has also been characterized in the form of somatostatin which inhibits the secretion of growth hormone (GH). In 1982, human pancreatic GH releasing factors were isolated from extracts of human pancreatic tumors, purified, characterized, synthesized and tested, which were found to promote the release of GH by the pituitary. The 44-residue peptide was subsequently found to have the same structure as human hypothalamic GRF and is herein referred to as hGRF. Each of these hypophysiotropic factors has been reproduced by total synthesis, and analogs of the native structures have been synthesized.

SUMMARY OF THE INVENTION

A polypeptide has now been isolated from rat hypothalamic extracts, purified, characterized, synthesized and tested which releases GH from cultured pituitary cells. The sequence of this 43-residue naturally occurring peptide is believed to be as follows: H--His--Ala--Asp--Ala--Ile--Phe--Thr--Ser--Ser--Tyr--Arg--Arg--Ile--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--His--Glu--Ile--Met--Asn--Arg--Gln--Gln--Gly--Glu--Arg--Asn--Gln--Glu--Gln--Arg--Ser--Arg--Phe--Asn--OH. The peptide is believed to be and is hereinafter referred to as rGRF (for rat hypothalamic GH releasing factor).

Pharmaceutical compositions in accordance with the invention include GRF analogs or a nontoxic salt thereof dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier. Such pharmaceutical compositions can be used in clinical medicine, both human and veterinary, for administration for therapeutic purposes, and also diagnostically. Moreover, they can be used to promote the growth of warm-blooded nonhuman animals, including fowl, and in aquiculture for cold-blooded animals, e.g. fish, eels, etc.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The nomenclature used to define the peptides is that specified by Schroder & Lubke, "The Peptides", Academic Press (1965), wherein in accordance with conventional representation the amino group at the N-terminus appears to the left and the carboxyl group at the C-terminus to the right. Where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

The invention provides synthetic GRF peptides having the following formula: H--R₁ --Ala--Asp--Ala--Ile--Phe--Thr--R₈ --Ser--R₁₀ --Arg--R₁₂ --R₁₃ --Leu--R₁₅ --Gln--Leu--R₁₈ --Ala--Arg--Lys--Leu--Leu--R₂₄ --R₂₅ --Ile--R₂₇ --R₂₈ --Arg--Gln--Gln--Gly--Glu--R₃₄ --Asn--Gln--Glu--R₃₈ --R₃₉ --R₄₀ -- Arg--R₄₂ --R₄₃ --Leu--Y wherein R₁ is Tyr, Met, Leu, D--Tyr, D--His or His; R₈ is Ser or Asn; R₁₀ is Tyr or D--Tyr; R₁₂ is Arg or Lys; R₁₃ is Ile or Val; R₁₅ is Gly or D--Ala; R₁₈ is Tyr or Ser; R₂₄ is His or Gln; R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile, Leu or Val; R₂₈ is Asn or Ser; R₃₄ is Arg or Ser; R₃₈ is Gln or Arg; R₃₉ is Arg or Gly; R₄₀ is Ser or Ala; R₄₂ is Phe or Ala; R₄₃ is Asn or Arg; and Y represents the carboxyl moiety of the amino acid residue at the C-terminus and is the radical --COOR,--CRO,--CONHNHR, --CON(R)(R') or --CH₂ OR, with R and R' being lower alkyl, fluoro lower alkyl or hydrogen, provided however that at least two but less than all of the following residues are present: R₈ is Asn, R₁₂ is Lys, R₁₃ is Val, R₁₈ is Ser, R₂₄ is Gln, R₂₅ is Asp, R₂₈ is Ser, or a fragment thereof extending from the N-terminus in a continuous sequence at least to the residue in position 27, which fragment is biologically active to cause the release of GH from the pituitary, or a nontoxic salt of such peptide or peptide fragment. Preferably there is an amide or lower alkyl amide at the C-terminus, i.e. Y is COHN₂ or CONHR. Methyl, ethyl and propyl are the preferred lower alkyl groups. When Met appears in position 1, it may be preferable to have another residue in position 27.

Fragments of the foregoing peptides also have biological potency, and such biologically active fragments are considered as falling within the scope of the overall invention. It is felt that the peptide should extend from the N-terminus at least through residue-27. When the peptide fragment extends only to residue 27 or 28, Y is NH₂ or a substituted amide. When the peptide has 40 or more residues, there is no clear preference for the moiety at the C-terminus.

The peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation, by classical solution couplings, or by the employment of recently developed recombinant DNA techniques for rGRF and those analogs containing only natural amino acid residues. For example, the techniques of exclusively solid-phase synthesis are set forth in the textbook "Solid-Phase Peptide Synthesis", Stewart & Young, Freeman & Co., San Francisco, 1969, and are exemplified by the disclosure of U.S. Pat. No. 4,105,603, issued Aug. 8, 1978 to Vale et al. Classical solution synthesis is described in detail in the treatise "Methoden der Organischen Chemie (Houben-Wey): Synthese von Peptiden", E. Wunsch (editor) (1974) Georg Thieme Verlag, Stuttgart, W. Ger. The fragment condensation method of synthesis is exemplified in U.S. Pat. No. 3,972,859 (Aug. 3, 1976). Other available syntheses are exemplified by U.S. Pat. No. 3,842,067 (Oct. 15, 1974) and U.S. Pat. No. 3,862,925 (Jan. 28, 1975).

Common to such syntheses is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the group is ultimately removed. Usually also common is the protection of an alpha-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in the synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with side-chain protecting groups linked to the appropriate residues.

Also considered to be within the scope of the present invention are intermediates of the formula: X¹ --R₁ (X or X²)--Ala--Asp(X³)--Ala--Ile--Phe--Thr(X⁴)--R₈ (X⁴ or X⁵)--Ser(X⁴)--R₁₀ (X²)--Arg(X⁶)--R₁₂ (X⁶ or X⁷)--R₁₃ --Leu--R₁₅ --Gln(X⁵)--Leu--R₁₈ (X² or X⁴)--Ala--Arg(X⁶)--Lys(X⁷)--Leu--Leu--R₂₄ (X or X⁵)--R₂₅ (X³)--Ile--R₂₇ --R₂₈ (X⁴ or X⁵)--Arg(X⁶)--Gln(X⁵)--Gln(X⁵)--Gly--Glu(X³)--R.sub.34 (X⁴ or X⁶)--Asn(X⁵)--Gln(X⁵)--Glu(X³)--R₃₈ (X⁵ or X⁶)--R₃₉)X⁶)--R₄₀ (X⁴)--Arg(X⁶)--R₄₂ --R₄₃ (X⁵ or X⁶)--Leu--X wherein: X¹ is either hydrogen or an α-amino protecting group. The α-amino protecting groups contemplated by X¹ are those well known to be useful in the art of step-wise synthesis of polypeptides. Among the classes of α-amino protecting groups which may be employed as X¹ are (1) aromatic urethan-type protecting groups, such as fluorenylmethyloxycarbonyl (FMOC), benzyloxycarbonyl (Z) and substituted Z, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl; (2) aliphatic urethan protecting groups, such as t-butyloxycarbonyl (BOC), diisopropylmethyloxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; and (3)cycloalkyl urethan-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl. The preferred α-amino protecting group is BOC.

X is hydrogen or a protecting group for the imidazole nitrogen of His, such as Tos.

X² may be a suitable protecting group for the phenolic hydroxyl group of Tyr, such as tetrahydropyranyl, tert-butyl, trityl, Bzl, CBZ, 4Br--CBZ and 2,6-dichlorobenzyl (DCB). The preferred protecting group is 2,6-dichlorobenzyl. X² can be hydrogen which means that there is no side-chain protecting group on the amino acid residue in that position.

X³ is hydrogen or a suitable ester-forming protecting group for the carboxyl group of Asp or Glu, such as benzyl (OBzl), 2,6-dichlorobenzyl, methyl and ethyl.

X⁴ may be a suitable protecting group for the hydroxyl group of Thr or Ser, such as acetyl, benzoyl, tert-butyl, trityl, tetrahydropyranyl, Bzl, 2,6-dichlorobenzyl and CBZ. The preferred protecting group is Bzl. X⁴ can be hydrogen, which means there is no protecting group on the hydroxyl group.

X⁵ is hydrogen or a suitable protecting group for the side chain amido group of Asn or Gln. It is preferably xanthyl (Xan).

X⁶ is a suitable protecting group for the guanidino group of Arg, such as nitro, Tos, CBZ, adamantyloxycarbonyl, and BOC, or is hydrogen.

X⁷ is hydrogen or a suitable protecting group for the side chain amino group of Lys. Illustrative of suitable side chain amino protecting groups are 2-chlorobenzyloxycarbonyl (2-Cl--Z), Tos, t-amyloxycarbonyl and BOC.

Met can optionally be protected by oxygen, but is preferably left unprotected.

The selection of a side chain amino protecting group is not critical except that generally one is chosen which is not removed during deprotection of the α-amino groups during the synthesis. However, for some amino acids, e.g. His, Asn and Gln, protection is not generally necessary after coupling is completed, and the protecting groups may be the same.

X⁹ is a suitable protecting group for the C-terminal carboxyl group, such as the ester-forming group X³, or is an anchoring bond used in solid-phase synthesis for linking to a solid resin support, or is des-X⁹, in which case the residue at the C-terminal has a carboxyl moiety which is Y, as defined hereinbefore. When a solid resin support is used, it may be any of those known in the art; in which case X⁹ may have one of the following formulae: --O--CH₂ --resin support, --N-Alkylaminomethyl (NAAM) resin support, --NH--benzhydrylamine (BHA) resin support or --NH--paramethylbenzhydrylamine (MBHA) resin support. When the unsubstituted amide is desired, use of BHA or MBHA resin is preferred because HF or strong acid cleavage directly gives the amide. In case the N-methyl amide is desired, it can be generated from an N-methyl BHA resin. Should other substituted amides be desired, use of the NAAM resins are preferred. Should other groups than the free acid be desired at the C-terminus, it may be preferable to synthesize the peptide using classical methods as set forth in the Houben-Weyl text.

In the formula for the intermediate, at least one of the X-groups is a protecting group or X⁹ includes resin support. Thus, the invention also provides a method for manufacturing a peptide of interest by (a) forming a peptide having at least one protective group and the formula (II): X¹ --R₁ (X or X²)--Ala-Asp(X³)--Ala--Ile--Phe--Thr(X⁴)--R₈ (X⁴ or X⁵)--Ser(X⁴)--Tyr(X²)--Arg(X⁶)--R₁₂ (X⁶ or X⁷)--R₁₃ --Leu-Gly-Gln(X⁵)--Leu--R₁₈ (X² or X⁴)--Ala-Arg(X⁶)--Lys(X⁷)--Leu--Leu--R₂₄ (X or X⁵)--R₂₅ (X³)--Ile--R₂₇ --R₂₈ (X⁴ or X⁵)--Arg(X⁶)--Gln(X⁵)--Gln(X⁵)--Gly--Glu(X³)--R.sub.34 (X⁴ or X⁶)--Asn(X⁵)--Gln(X⁵)--Glu(X³)--R₃₈ (X⁵ or X⁶)--R₃₉ (X⁶)--R₄₀ (X⁴)--Arg(X⁶)-- R₄₂ --R₄₃ (X⁵ or X⁶)--Leu--X⁹ wherein: X, X¹, X², X³, X⁴, X⁵, X⁶ and X⁷ are each either hydrogen or a protective group and X⁹ is either a protective group or an anchoring bond to resin support or des-X⁹, in which case the residue at the C-terminal has a carboxy moiety which is Y; (b) splitting off the protective group or groups or anchoring bond from said peptide of the formula (II); and (c) if desired, converting a resulting peptide into a nontoxic salt thereof.

In selecting a particular side chain protecting group to be used in the synthesis of the peptides, the following general rules are followed: (a) the protecting group preferably retains its protecting properties and is not be split off under coupling conditions, (b) the protecting group should be stable to the reagent and, with the exception of Xan, is preferably stable under the reaction conditions selected for removing the α-amino protecting group at each step of the synthesis, and (c) the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.

When peptides are not prepared using recombinant DNA technology, they are preferably prepared using solid phase synthesis, such as that generally described by Merrifield, J. Am. Chem. Soc., 85, p. 2149 (1963), although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminal end of the peptide by coupling a protected α-amino acid to a suitable resin. Such a starting material can be prepared by attaching an α-amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a BHA resin or MBHA resin. The preparation of the hydroxymethyl resin is described by Bodansky et al., Chem. Ind. (London) 38, 1597-1598 (1966). Chloromethylated resins are commercially available from Bio Rad Laboratories, Richmond, California and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart et al., "Solid Phase Peptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, pp. 1-6. BHA and MBHA resin supports are commercially available and are generally used only when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminal.

The C-terminal amino acid, i.e. Leu, protected by BOC, can be first coupled to the chloromethylated resin according to the procedure set forth in Chemistry Letters, K. Horiki et al. 165-168 (1978), using KF in DMF at about 80° C. for 24 hours with stirring, when the 43-residue peptide is to be synthesized. Following the coupling of the BOC-protected amino acid to the resin support, the α-amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride, TFA alone or HCl in dioxane. The deprotection is carried out at a temperature between about 0° C. and room temperature. Other standard cleaving reagents and conditions for removal of specific α-amino protecting groups may be used as described in Schroder & Lubke, "The Peptides", 1 pp. 72-75 (Academic Press 1965).

After removal of the α-amino protecting group, the remaining α-amino- and side chain-protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor. The selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,N'-dicyclohexyl carbodiimide (DCCI).

The activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art. Examples of suitable activating reagents are carbodiimides, such as N,N'-diisopropyl carbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activating reagents and their use in peptide coupling are described by Schroder & Lubke supra, in Chapter III and by Kapoor, J. Phar. Sci., 59, pp. 1-27 (1970).

Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in about a fourfold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF): CH₂ Cl₂ (1:1) or in DMF or CH₂ Cl₂ alone. In cases where incomplete coupling occurs, the coupling procedure is repeated before removal of the α-amino protecting group prior to the coupling of the next amino acid. The success of the coupling reaction at each stage of the synthesis, if performed manually, is preferably monitored by the ninhydrin reaction, as described by E. Kaiser et al., Anal. Biochem. 34, 595 (1970). The coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. Biopolymers, 1978, 17, pp. 1927-1938.

After the desired amino acid sequence has been completed, the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride, which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups X, X², X³, X⁴, X⁵, X⁶ and X⁷, the anchoring bond X⁹ and the α-amino protecting group X¹, to obtain the peptide in the form of the free acid. If Met is present in the sequence, the BOC protecting group is preferably first removed using trifluoroacetic acid(TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S-alkylation. When using hydrogen fluoride for cleaving, anisole and methylethyl sulfide are included in the reaction vessel for scavenging.

As one approach for preparing the synthetic peptides using recombinant DNA technology, a Met residue is preferably included as an additional residue at the N-terminus, and Met should not be present elsewhere in the peptide. Likewise, all of the residues employed in the peptide must be naturally occurring L-isomer amino acids or Gly. As a result, a preferred peptide intermediate composition is produced having the formula: H--Met--R₁ --Ala--Asp--Ala--Ile--Phe--Thr--R₈ --Ser--Try--Arg--R₁₂ --R₁₃ --Leu--Gly--Gln--Leu--R₁₈ --Ala--Arg--Lys--Leu--Leu--R₂₄ --R₂₅ --Ile--R₂₇ --R₂₈ --Arg--Gln--Gln--Gly--Glu--R₃₄ --Asn--Gln--Glu--R₃₈ --R₃₉ --R₄₀ --Arg--R₄₂ --R₄₃ --Leu--Y wherein R₁ is Tyr, Leu or His; R₈ is Ser or Asn; R₁₂ is Arg or Lys; R₁₃ is Ile or Val; R₁₈ is Tyr or Ser; R₂₄ is His or Gln; R₂₅ is Glu or Asp; R₂₇ is Ala, Nle, Ile, Leu or Val; R₂₈ is Asn or Ser; R₃₄ is Arg or Ser; R₃₈ is Gln or Arg; R₃₉ is Arg or Gly; R₄₀ is Ser or Ala; R₄₂ is Phe or Ala; R₄₃ is Asn or Arg; wherein Y represents the carboxyl moiety of the C-terminus residue and is either --COOH or CONH₂.

Once this peptide is expressed by a genetically modified microorganism and the intermediate composition is recovered and purified using known methods, the intermediate is treated with cyanogen bromide to cleave the Met residue at the N-terminus and produce the biologically active peptide. Although the formula set forth above indicates that the peptide will extend through position 44, as indicated elsewhere herein, substantial biological activity is provided by peptides having as few as 27 to 29 residues, and such shorter fragments are considered to be generally equivalent for many purposes and to be included within the scope of the preferred intermediate that is provided by the invention which is intended for production by recombinant DNA technology.

The following Example set forth the preferred method for synthesizing GRF analogs by the solid-phase technique. It will of course be appreciated that the synthesis of correspondingly shorter or longer peptides can be effected in the same manner by eliminating or introducing the requisite number of amino acids at the C-terminus.

EXAMPLE I

The synthesis of [Nle²⁷,Asn²⁸ ]-hGRF(1-29)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Asn--Arg--NH₂ is conducted in a stepwise manner using a Beckman 990 peptide synthesizer on an MBHA resin having a substitution range of about 0.1 to 0.5 mmoles/g. resin. Coupling of BOC--Arg(Tos) to the resin is performed by the general procedure set forth in Vale et al. U.S. Pat. No. 4,292,313 using a mixture of methylene chloride and DMF at about 60° C. for 24 hours with stirring, and it results in the substitution of about 0.35 mmol. Arg per gram of resin.

After deblocking and neutralization, the peptide chain is built step-by-step on the resin. Deblocking, neutralization and addition of each amino acid is performed in general accordance with the procedure set forth in detail in Rivier, J, J. Amer. Chem. Soc., 96, 2986-2992 (1974). All solvents that are used are carefully degassed by sparging with an inert gas, e.g. helium or nitrogen.

Deblocking is preferably carried out in accordance with Schedule A which follows:

                  SCHEDULE A                                                       ______________________________________                                         Reagent             Mixing time (Min.)                                         ______________________________________                                         1.    60% TFA/2% ethanedithiol                                                                         10                                                     2.    60% TFA/2% ethanedithiol                                                                         15                                                     3.    IPA/1% ethanedithiol                                                                             0.5                                                    4.    Et.sub.3 N (10%) in CH.sub.2 Cl.sub.2                                                            0.5                                                    5.    MeOH              0.5                                                    6.    Et.sub.3 N (10%) in CH.sub.2 Cl.sub.2                                                            0.5                                                    7.    MeOH (twice)      0.5                                                    8.    CH.sub.2 Cl.sub.2 (twice)                                                                        0.5                                                    ______________________________________                                    

The couplings are preferably carried out as set out in Schedule B which follows:

                  SCHEDULE B                                                       ______________________________________                                         Reagent           Mixing time (Min.)                                           ______________________________________                                         9.     DCCI           --                                                       10.    Boc-amino acid 50-90                                                    11.    MeOH (twice)   0.5                                                      12.    CH.sub.2 Cl.sub.2 (twice)                                                                     0.5                                                      13.    Ac.sub.2 O (3M) in CH.sub.2 Cl.sub.2                                                          15.0                                                     14.    CH.sub.2 Cl.sub.2                                                                             0.5                                                      15.    MeOH           0.5                                                      16.    CH.sub.2 Cl.sub.2 (twice)                                                                     0.5                                                      ______________________________________                                    

Briefly, one to two mmol. of BOC-protected amino acid in methylene chloride is used per gram of resin, plus one equivalent of 1.0 molar DCCI in methylene chloride for two hours. As mentioned before, when BOC-Arg(Tos) is being coupled, a mixture of 50% DMF and methylene chloride is used. Bzl ether is used as the hydroxy side-chain protecting group for Ser and Thr. The amido group of Asn or Gln is protected by Xan when DCC coupling is used, as is preferred. P-nitrophenyl ester(ONp) may also be used to activate the carboxyl end of Asn or Gln, and for example, BOC--Asn(ONp) can be coupled overnight using one equivalent of HObt in a 50% mixture of DMF and methylene chloride, in which case no DCC is added. 2-chloro-benzyloxycarbonyl(2Cl-Z) is used as the protecting group for the Lys side chain. Tos is used to protect the guanidino group of Arg and the imidazole nitrogen of His (if present, and the Glu or Asp carboxyl group is protected as the Bzl ester (OBzl). The phenolic hydroxyl group of Tyr is protected with 2,6--dichlorobenzyl (DCB). At the end of the synthesis, the following composition is obtained: BOC--Tyr(X²)--Ala--Asp(X³)--Ala--Ile--Phe--Thr(X⁴)--Asn(X.sup.5)--Ser(X⁴)--Tyr(X²)--Arg(X⁶)--Lys(X⁷)--Val--Leu--Gly--Gln(X⁵)--Leu--Ser(X⁴)--Ala--Arg(X⁶)--Lys(X⁷)--Leu--Leu--Gln(X⁵)--Asp(X³)--Ile--Nle--Asn(X⁵)-NH-resin support wherein X² is DCB, X³ is OBzl, X⁴ is Bzl, X⁵ is Xan, X⁶ is Tos and X⁷ is 2Cl--Z. Xan may have been partially or totally removed by TFA treatment used to deblock the α-amino protecting group.

After the final Tyr residue has been coupled to the resin, BOC is removed with 60% TFA in CH₂ Cl₂. In order to cleave and deprotect the remaining protected peptide-resin, it is treated with 1.5 ml. anisole, 0.5 ml. methylethylsulfide and 15 ml. hydrogen fluoride (HF) per gram of peptide-resin, at -20° C. for one-half hour and at 0.° C. for one-half hour. After elimination of the HF under high vacuum, the resin-peptide remainder is washed alternately with dry diethyl ether and chloroform, and the peptide is then extracted with degassed 2 N aqueous acetic acid and separated from the resin by filtration.

The cleaved and deprotected peptide is then dissolved in 0-5% acetic acid and subjected to purification which may include Sephadex G-50 fine gel filtration.

The peptide is then further purified by preparative or semi-preparative HPLC as described in Rivier et al., Peptides: Structure and Biological Function, (1979) pp. 125-8 and Marki et al. J. Am. Chem. Soc. 103, 3178 (1981). Cartridges fitting Waters Associates prep LC-500 are packed with 15-20 C₁₈ Silica from Vydac (300A). A gradient of CH₃ CN in TEAP is generated by a low pressure Eldex gradient maker, as described in Rivier, J., J. Liq. Chromatography 1, 343-367 (1978). The chromatographic fractions are carefully monitored by HPLC, and only the fractions showing substantial purity are pooled. Desalting of the purified fractions, independently checked for purity, is achieved using a gradient of CH₃ CN in 0.1% TFA. The center cut is then lyophilized to yield the desired peptide, the purity of which can be greater than 98%.

EXAMPLE II

The synthesis of [Asn²⁸ ]-hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Asn--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu---Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as generally described in Vale et al. U.S. Pat. No. 4,292,313. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE III

The synthesis of [Asn²⁸ ]-hGRF(1-32)--NHEt having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Asn--Arg--Gln--Gln--Gly--NHCH₂ CH₃ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an N-ethylaminomethyl resin, in the manner generally described in "Peptide N-Alkylamides by Solid Phase Synthesis", Kornreich, W. et al., J. of Intl. Protein Chemistry (1985) and in copending U.S. application Ser. No. 545,077, filed Oct. 24, 1983. The peptide is judged to be substantially pure asking TLC and HPLC.

EXAMPLE IV

The synthesis of [Ser⁸ ]--hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Ser--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. This analog is judged to be substantially pure using TLC and HPLC.

EXAMPLE V

The synthesis of [Arg³⁴ ]--hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Arg--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

The synthesis is repeated substituting His for Tyr at the N-terminus to produce [His¹,Arg³⁴ ]-hGRF (1-44)--NH₂.

EXAMPLE VI

The synthesis of [Gln³⁸,Arg³⁹ ]-hGRF(1-44)--OH having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Gln--Arg--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on a chloromethylated resin as set forth in Chemistry Letters, supra. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE VII

The synthesis of [Arg¹² ]-hGRF(1-32)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Arg--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE VIII

The synthesis of [Glu²⁵, Nle²⁷ ]-gGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Lew--Leu--Gln--Glu--Ile--Nle--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE IX

The synthesis of [Ile¹³,Ser⁴⁰ ]--hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Ile--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ser--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC aND HPLC.

EXAMPLE X

The synthesis of [His²⁴ ]--hGRF(1-40)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--His--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XI

The synthesis of [Tyr¹⁸ ]--hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC aND HPLC.

EXAMPLE XII

The synthesis of [Phe⁴² ]--hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Phe--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XIII

The synthesis of [Arg³⁹, Asn⁴³ ]-hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Arg--Ala--Arg--Ala--Asn--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XIV

The synthesis of [Arg¹²,Asn²⁸ ]--hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Arg--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Asn--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesize on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XV

The synthesis of [Tyr¹⁸, Nle²⁷,Arg³⁹ ]--hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Arg--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XVI

The synthesis of [Ser⁸,Nle²⁷,]--hGRF(1-32)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Ser--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Ser--Arg--Gln--Gln--Gly--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XVII

The synthesis of [Tyr¹⁸, His²⁴, Nle²⁷ ]-hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--His--Asp--Ile--Nle--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XVIII

The synthesis of [Ile¹³,Tyr¹⁸,Nle²⁷ ]-hGRF(1-29)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Ile--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Ser--Arg--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XIX

The synthesis of ]Arg¹²,Nle²⁷ ]--hGRF(1-32)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Arg--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Ser--Arg--Gln--Gln--Gly--NH₂ is conducted in a stepwise manner using a beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XX

The synthesis of [His¹,Tyr¹⁸,Nle²⁷ ]-hGRF(1-29)--NH₂ having the formula: H--His--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Ser--Arg--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXI

The synthesis of [Nle²⁷,Asn²⁸,Ser⁴⁰ ]-hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Asn--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ser--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXII

The synthesis of [His²⁴,Glu²⁵,Nle²⁷,]-hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--His--Glu--Ile--Nle--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXIII

The synthesis of [D-Tyr¹⁰,Tyr¹⁸,Arg³⁴ ]-hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--D--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--Arg--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXIV

The synthesis of [Ile¹³,Glu²⁵,Nle²⁷ ]-hGRF(1-27)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Ile--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Glu--Ile--Nle--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXVI

The synthesis of [His²⁴,Nle²⁷,Asn²⁸ ]-hGRF(1-29)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--His--Asp--Ile--Nle--Asn--Arg--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXVI

The synthesis of [D--Tyr¹⁰,Nle²⁷,Asn²⁸ ]-hGRF(1-32)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--D--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Asn--Arg--Gln--Gln--Gly--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXVII

The synthesis of [Tyr¹,Lys¹²,Ser²⁸ ]-rGRF(1-29)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Ser--Ser--Tyr--Arg--Lys--Ile--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--His--Glu--Ile--Met--Ser--Arg--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXIII

The synthesis of [Ser⁸,His²⁴,Nle²⁷,Asn²⁸ ]-hGRF(1-32)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Ser--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--His--Asp--Ile--Nle--Asn--Arg--Gln--Gln--Gly--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

EXAMPLE XXIX

The synthesis of [Arg¹²,His²⁴,Glu²⁵,Nle²⁷ ]-hGRF(1-44)--NH₂ having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Arg--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--His--Glu--Ile--Nle--Ser--Arg--Gln--Gln--Gly--Glu--Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer on an MBHA resin as in Example I. The peptide is judged to be substantially pure using TLC and HPLC.

Various of the synthetic peptides prepared in the Examples are compared with synthetic hpGRF(1-40)--OH in in vitro assays and are found to exhibit generally greater potencies for the secretion of GH and similar intrinsic activities.

To determine the effectiveness of the various synthetic peptides to promote the release of growth hormone, in vitro assays are carried out using synthetic hpGRF(1-40)--OH as a standard in side-by-side comparison with equimolar concentrations of the various other analogs and fragments synthesized. Cultures are used which include cells of rat pituitary glands removed some three to five days previously. Cultures which are considered optimal for the secretion of growth hormone are used for the comparative testing, in the general manner described in Vale et al. Endocrinology, 91, 562-572 (1972) and as more particularly described in Vale et al. Endocrinology, (1983), in press. Incubation with the substance to be tested is carried out for 3 to 4 hours, and aliquots of the culture medium are removed and processed to measure their contents in immunoreactive GH(ir GH) by a well-characterized radioimmunoassay.

The results of this comparative testing for equimolar concentrations are shown in Table I.

                  TABLE I                                                          ______________________________________                                         Peptide                Comparison %                                            ______________________________________                                         npGRF(1-40)-OH         100%                                                    (standard for this test)                                                       [Tyr.sup.1,Lys.sup.12,Ser.sup.28 ]--rGRF(1-29)-NH.sub.2                                               %                                                       [D-Tyr.sup.10,Nle.sup.27,Asn.sup.28 ]-hGRF(1-32)-NH.sub.2                                             %                                                       [His.sup.1,Tyr.sup.18,Nle.sup.27 ]--hGRF(1-29)-NH.sub.2                                               %                                                       [Nle.sup.27,Asn.sup.28 ]--hGRF(1-29)-NH.sub.2                                                         520%                                                    [Arg.sup.12,Nle.sup.27 ]--hGRF(1-32)-NH.sub.2                                                         %                                                       [His.sup.1,Arg.sup.12,Asn.sup.28 ]--hGRF(1-29)-NH.sub.2                                               %                                                       [Asn.sup.28 ]--hGRF-(1-32)-NH.sub.2                                                                   %                                                       ______________________________________                                    

In vitro testing of these synthetic peptides shows that the EC₅₀ varies from 20-100 picomolar and that the lowest effective concentration is 3-8 picomolar. The maximum effective concentration for rGRF(1-43)--OH is 1 nanomolar.

In addition to the in vitro tests for secretion of growth hormone, in vivo experiments are also run by injecting the synthetic peptide through an indwelling catheter into freely running normal male rats. Animals are pretreated with FLA-63, a dopamine hydroxylase inhibitor that suppresses spontaneous GH secretion without affecting the response to exogenous GRF. Blood samples are taken through the same catheter immediately prior to and 5 and 20 minutes after injections; GH levels in blood are measured by radioimmunoassay. The results show that synthetic rGRF(1-43)--OH and analogs and fragments thereof are powerful stimulators of the secretion of pituitary GH. Dosages between about 40 nanograms and about 25 micrograms per Kg. of body weight are considered to be effective in causing secretion.

Synthetic rGRF should be useful for human applications in which a physician wishes to elevate GH production. Stimulation of GH secretion by rGRF and analogs is of interest in patients with complete or relative GH deficiency caused by underproduction of endogenous GRF. Furthermore, it is probable that increased GH secretion and its attendant increase in growth could be obtained in humans or animals with normal GH levels. Moreover, rGRF administration should alter body fat content and modify other GH-dependent metabolic, immunologic and developmental processes. For example, rGRF may be useful as a means of stimulating anabolic processes in human beings under circumstances such as following the incurring of burns. As another example, rGRF may be administered to commercial warm-blooded animals, such as chickens, turkeys, pigs, goats, cattle and sheep, and may be used in aquiculture for raising fish and other cold-blooded marine animals, e.g. sea turtles, shrimp and eels, and amphibians, to accelerate growth and increase the ratio of protein to fat gained by feeding effective amounts of the peptides.

For administration to humans, synthetic rGRF peptides should have a purity of at least about 93% and preferably at least 98%. This purity generally means the intended peptide constitutes the stated weight % of all peptides and peptide fragments present. For the administration of synthetic rGRF peptides to commercial and other animals in order to promote growth and reduce fat content, a purity as low as about 5%, or even as low as 0.001%, may be acceptable. If produced by genetic engineering methods, synthetic peptides may have such very low purities; however these purities would still be higher than those found in initial natural extracts.

Synthetic rGRF or the nontoxic salts thereof, combined with a pharmaceutically or veterinarily acceptable carrier to form a pharmaceutical composition, may be administered to animals, including humans, either intravenously, subcutaneously, intramuscularly, percutaneously, e.g. intranasally or even orally. The administration may be employed by a physician to stimulate the release of GH where the host being treated requires such therapeutic treatment. The required dosage will vary with the particular condition being treated, with the severity of the condition and with the duration of desired treatment.

Such peptides are often administered in the form of nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be orally administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

The peptides should be administered to humans under the guidance of a physician, and pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Usually, the parenteral dosage will be from about 40 nanograms to about 25 micrograms of the peptide per kilogram of the body weight of the host.

Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto. For example, modifications in the peptide chain, particularly deletions beginning at the carboxyl terminal of the peptide, can be made in accordance with the known experimental practises to date to create peptides or peptide fragments that retain all or very substantial portions of the biological potency of the peptide, and such peptides are considered as being within the scope of the invention. Moreover, additions may be made to the C-terminus and/or generally equivalent residues can be substituted for naturally occurring residues, as is well-known in the overall art of peptide chemistry, to produce analogs having at least a substantial portion of the potency of the native polypeptide without deviating from the scope of the invention.

Various features of the invention are emphasized in the claims which follow. 

What is claimed is:
 1. A synthetic peptide having the formula: H--R₁ --Ala--Asp--Ala--Ile--Phe--Thr--R₈ --Ser--R₁₀ --Arg--R₁₂ --R₁₃ --Leu--R₁₅ --Gln--Leu--R₁₈ --Ala--Arg--Lys--Leu--Leu--R₂₄ --R₂₅ --Ile--R₂₇ --R₂₈ --Arg--Gln--Gln--Gly--Glu--R₃₄ --Asn--Gln--Glu--R₃₈ --R₃₉ --R₄₀ --Arg--R₄₂ --R₄₃ --Leu--Y wherein R₁ is Tyr, Met, Leu, D--Tyr, D--His or His; R₈ is Ser or Asn; R₁₀ is Tyr or D--Tyr; R₁₂ is Arg or Lys; R₁₃ is Ile or Val; R₁₅ is Gly or D--Ala; R₁₈ is Tyr or Ser; R₂₄ is His or Gln; R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile, Leu or Val; R₂₈ is Asn or Ser; R₃₄ is Arg or Ser; R₃₈ is Gln or Arg; R₃₉ is Arg or Gly; R₄₀ is Ser or Ala; R₄₂ is Phe or Ala; R₄₃ is Asn or Arg; and Y represents the carboxyl moiety of the amino acid residue at the C-terminus and is the radical --COOR, --CRO, --CONHNHR, --CON(R)(R') or --CH₂ OR, with R and R' being lower alkyl, fluoro lower alkyl or hydrogen, provided however that at least two but not more than six of the following residues are present: R₈ is Asn, R₁₂ is Lys, R₁₃ is Val, R₁₈ is Ser, R₂₄ is Gln, R₂₅ is Asp, R₂₈ is Ser, or a fragment thereof extending from the N-terminus in a continuous sequence at least to the residue in position 29, which fragment is biologically active to cause the release of GH from the pituitary, or a pharmaceutically acceptable nontoxic salt of such peptide or peptide fragment.
 2. The peptide of claim 1 wherein Y is CONH₂ or CONHR.
 3. The peptide of claim 2 wherein R₂₈ is Asn.
 4. The peptide of claim 3 wherein R₂₇ is Nle.
 5. The peptide of claim 3 wherein R₁ is His.
 6. The peptide of claim 5 wherein R₁₂ is Arg.
 7. The peptide of claim 1 wherein R₂₇ is Nle.
 8. The peptide of claim 7 wherein R₁₃ is Ile.
 9. The peptide of claim 7 wherein R₁₈ is Tyr.
 10. The peptide of claim 1 wherein R₈ is Ser.
 11. The peptide of claim 1 wherein R₁₈ is Tyr.
 12. The peptide of claim 1 wherein R₂₄ is His.
 13. The peptide of claim 12 wherein R₂₇ is Nle.
 14. The peptide of claim 1 wherein R₁ is Tyr, R₈ is Asn, R₁₀ is Tyr, R₁₂ is Lys, R₁₃ is Val, R₁₅ is Gly, R₁₈ is Ser, R₂₄ is Gln, R₂₅ is Asp, R₂₇ is Nle, R₂₈ is Asn and Y is --CONH₂ or --CONHR.
 15. The peptide of claim 1 having the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Tyr--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Asn--Arg--NH₂.
 16. A pharmaceutical composition for stimulating the release of GH in an animal comprising an effective amount of the peptide of claim 1 or a pharmaceutically acceptable nontoxic salt thereof, and a pharmaceutically or veterinarily acceptable liquid or solid carrier therefor.
 17. A method of stimulating the release of growth hormone in an animal, which comprises administering to said animal an effective amount of a synthetic peptide having the formula: H--R₁ --Ala--Asp--Ala--Ile--Phe--Thr--R₈ --Ser--R₁₀ --Arg--R₁₂ --R₁₃ --Leu--R₁₅ --Gln--Leu--R₁₈ --Ala--Arg--Lys--Leu--Leu--R₂₄ --R₂₅ --Ile--R₂₇ --R₂₈ --Arg--Gln--Gln--Gly--Glu--R₃₄ --Asn--Gln--Glu--R₃₈ --R₃₉ --R₄₀ --Arg--R₄₂ --R₄₃ --Leu--Y wherein R₁ is Tyr, Met, Leu, D--Tyr, D--His or His; R₈ is Ser or Asn; R₁₀ is Tyr or D--Tyr; R₁₂ is Arg or Lys; R₁₃ is Ile or Val; R₁₅ is Gly or D--Ala; R₁₈ is Tyr or Ser; R₂₄ is His or Gln; R₂₅ is Glu or Asp; R₂₇ is Met, Ala, Nle, Ile, Leu or Val; R₂₈ is Asn or Ser; R₃₄ is Arg or Ser; R₃₈ is Gln or Arg; R₃₉ is Arg or Gly; R₄₀ is Ser or Ala; R₄₂ is Phe or Ala; R₄₃ is Asn or Arg; and Y represents the carboxyl moiety of the amino acid residue at the C-terminus and is the radical --COOR,--CRO,--CONHNHR, --CON(R)(R') or --CH₂ OR, with R and R' being lower alkyl, fluoro lower alkyl or hydrogen, provided however that at least two but not more than six of the following residues are present: R₈ is Asn, R₁₂ is Lys, R₁₃ is Val, R₁₈ is Ser, R₂₄ is Gln, R₂₅ is Asp, R₂₈ is Ser, or a fragment thereof extending from the N-terminus in a continuous sequence at least to the residue in position 29, which fragment is biologically active to cause the release of GH from the pituitary, or a pharmaceutically acceptable nontoxic salt of such peptide or peptide fragment.
 18. A method in accordance with claim 17 wherein R₁ is Tyr, R₈ is Asn, R₁₀ is Tyr, R₁₂ is Lys, R₁₃ is Val, R₁₅ is Gly, R₁₈ is Ser, R₂₄ is Gln, R₂₅ is Asp, R₂₇ is Nle, R₂₈ is Asn and Y is --CONH₂ or CONHR.
 19. A method in accordance with claim 17 wherein the peptide has the formula: H--Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--Leu--Gln--Asp--Ile--Nle--Asn--Arg--NH₂.
 20. A method for promotion of growth in warm-blooded nonhuman animals in accordance with claim
 17. 